The NO pathway acts late during the fertilization response in sea urchin eggs

Both the inositol 1,4,5-trisphosphate (InsP(3)) and ryanodine receptor pathways contribute to the Ca(2+) transient at fertilization in sea urchin eggs. To date, the precise contribution of each pathway has been difficult to ascertain. Evidence has accumulated to suggest that the InsP(3) receptor pathway has a primary role in causing Ca(2+) release and egg activation. However, this was recently called into question by a report implicating NO as the primary egg activator. In the present study we pursue the hypothesis that NO is a primary egg activator in sea urchin eggs and build on previous findings that an NO/cGMP/cyclic ADP-ribose (cADPR) pathway is active at fertilization in sea urchin eggs to define its role. Using a fluorescence indicator of NO levels, we have measured both NO and Ca(2+) at fertilization and establish that NO levels rise after, not before, the Ca(2+) wave is initiated and that this rise is Ca(2+)-dependent. By inhibiting the increase in NO at fertilization, we find not that the Ca(2+) transient is abolished but that the duration of the transient is significantly reduced. The latency and rise time of the transient are unaffected. This effect is mirrored by the inhibition of cGMP and cADPR signaling in sea urchin eggs at fertilization. We establish that cADPR is generated at fertilization, at a time comparable to the time of the rise in NO levels. We conclude that NO is unlikely to be a primary egg activator but, rather, acts after the initiation of the Ca(2+) wave to regulate the duration of the fertilization Ca(2+) transient.     

Increase of cGMP, cADP-ribose and inositol 1,4,5-trisphosphate preceding Ca(2+) transients in fertilization of sea urchin eggs.

Transient increases, or oscillations, of cytoplasmic free Ca(2+) concentration, [Ca(2+)](i), occur during fertilization of animal egg cells. In sea urchin eggs, the increased Ca(2+) is derived from intracellular stores, but the principal signaling and release system involved has not yet been agreed upon. Possible candidates are the inositol 1,4,5-trisphosphate receptor/channel (IP(3)R) and the ryanodine receptor/channel (RyR) which is activated by cGMP or cyclic ADP-ribose (cADPR). Thus, it seemed that direct measurements of the likely second messenger candidates during sea urchin fertilization would be essential to an understanding of the Ca(2+) signaling pathway. We therefore measured the cGMP, cADPR and inositol 1,4,5-trisphosphate (IP(3)) contents of sea urchin eggs during the early stages of fertilization and compared these with the [Ca(2+)](i) rise in the presence or absence of an inhibitor against soluble guanylate cyclase. We obtained three major experimental results: (1) cytosolic cGMP levels began to rise first, followed by cADPR and IP(3) levels, all almost doubling before the explosive increase of [Ca(2+)](i); (2) most of the rise in IP(3) occurred after the Ca(2+) peak; IP(3) production could also be induced by the artificial elevation of [Ca(2+)](i), suggesting the large increase in IP(3) is a consequence, rather than a cause, of the Ca(2+) transient; (3) the measured increase in cGMP was produced by the soluble guanylate cyclase of eggs, and inhibition of soluble guanylate cyclase of eggs diminished the production of both cADPR and IP(3) and the [Ca(2+)](i) increase without the delay of Ca(2+) transients. Taken together, these results suggest that the RyR pathway involving cGMP and cADPR is not solely responsible for the initiating event, but contributes to the Ca(2+) transients by stimulating IP(3) production during fertilization of sea urchin eggs.     

CA++ in fertilization and mitosis: the phosphatidylinositol cycle in sea urchin gametes and zygotes is involved in control of fertilization and mitosis

We determined that the phosphatidylinositol (PI) cycles in both sea urchin sperm and eggs are necessary for normal fertilization, and that the PI cycle in sea urchin zygotes is involved in control of mitosis. The PI cycle is involved in Ca++ homeostasis so our data are direct evidence that Ca++ is involved with control of mitosis and fertilization. We implicated the PI cycle by adding Li+ to sea urchin eggs, sperm, or zygotes: those effects of Li+ due to effects on the PI cycle were overcome by myo-inositol but not by its optical isomer, scyllitol, and not by mannitol.     

Function of a sea urchin egg Src family kinase in initiating Ca2+ release at fertilization

Egg activation at fertilization requires the release of Ca(2+) from the egg's endoplasmic reticulum, and recent evidence has indicated that a Src family kinase (SFK) may function in initiating this signaling pathway in echinoderm eggs. Here, we identify and characterize a SFK from the sea urchin Strongylocentrotus purpuratus, SpSFK1. SpSFK1 RNA is present in eggs, and an antibody made against a SpSFK1 peptide recognizes an approximately 58-kDa egg membrane-associated protein in eggs of S. purpuratus as well as another sea urchin Lytechinus variegatus. Injection of both species of sea urchin eggs with dominant-interfering Src homology 2 domains of SpSFK1 delays and reduces the release of Ca(2+) at fertilization. Injection of an antibody against SpSFK1 into S. purpuratus eggs also causes a small increase in the delay between sperm-egg fusion and Ca(2+) release. In contrast, when injected into eggs of L. variegatus, this same antibody has a dramatic stimulatory effect: it causes PLCgamma-dependent Ca(2+) release like that occurring at fertilization. Correspondingly, in lysates of L. variegatus eggs, but not S. purpuratus eggs, the antibody stimulates SFK activity. Injection of L. variegatus eggs with another antibody that recognizes the L. variegatus egg SFK also causes PLCgamma-dependent Ca(2+) release like that at fertilization. These results indicate that activation of a Src family kinase present in sea urchin eggs is necessary to cause Ca(2+) release at fertilization and is capable of stimulating Ca(2+) release in the unfertilized egg via PLCgamma, as at fertilization.     

Fertilization and nicotinic acid adenine dinucleotide phosphate induce pH changes in acidic Ca(2+) stores in sea urchin eggs

The second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) releases Ca(2+) from the acidic Ca(2+) stores of many organisms, including those of the sea urchin egg. We investigated whether the pH within the lumen of these acidic organelles changes in response to stimuli. Fertilization activates the egg by Ca(2+) release dependent upon NAADP, and accordingly, we report that fertilization also alters organellar pH in a spatio-temporally complex manner. Upon sperm fusion, vesicles deep in the egg center slowly acidify, whereas cortical vesicles undergo a rapid alkalinization. The cortical vesicle alkalinization is independent of exocytosis and cytosolic pH but coincides with the NAADP-dependent fertilization Ca(2+) wave. Microinjection of NAADP mimicked the fertilization cortical response, suggesting that it occurred within NAADP-sensitive acidic Ca(2+) stores. Our data show that NAADP and physiological stimuli alter the pH within intracellular organelles and suggest that NAADP signals through pH as well as Ca(2+).     

A functional genomic and proteomic perspective of sea urchin calcium signaling and egg activation

The sea urchin egg has a rich history of contributions to our understanding of fundamental questions of egg activation at fertilization. Within seconds of sperm-egg interaction, calcium is released from the egg endoplasmic reticulum, launching the zygote into the mitotic cell cycle and the developmental program. The sequence of the Strongylocentrotus purpuratus genome offers unique opportunities to apply functional genomic and proteomic approaches to investigate the repertoire and regulation of Ca(2+) signaling and homeostasis modules present in the egg and zygote. The sea urchin "calcium toolkit" as predicted by the genome is described. Emphasis is on the Ca(2+) signaling modules operating during egg activation, but the Ca(2+) signaling repertoire has ramifications for later developmental events and adult physiology as well. Presented here are the mechanisms that control the initial release of Ca(2+) at fertilization and additional signaling components predicted by the genome and found to be expressed and operating in eggs at fertilization. The initial release of Ca(2+) serves to coordinate egg activation, which is largely a phenomenon of post-translational modifications, especially dynamic protein phosphorylation. Functional proteomics can now be used to identify the phosphoproteome in general and specific kinase targets in particular. This approach is described along with findings to date. Key outstanding questions regarding the activation of the developmental program are framed in the context of what has been learned from the genome and how this knowledge can be applied to functional studies.     

Development of calcium releasing activity induced by inositol trisphosphate and cyclic ADP-ribose during in vitro maturation of sea urchin oocytes

During fertilization of sea urchin eggs, the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) transiently increases (Ca(2+) transient). Increased [Ca(2+)](i) results from a rapid release from intracellular stores, mediated by one or both of two signaling pathways; inositol 1,4,5-trisphosphate (IP(3)) and IP(3) receptor (IP(3)R) or cyclic GMP (cGMP), cyclic ADP-ribose (cADPR) and ryanodine receptor (RyR). During fertilization, cGMP and cADPR increase preceding the Ca(2+) transient, suggesting their contribution to this. If the RyR pathway contributed to the Ca(2+) transient, its Ca(2+) releasing activity would develop in parallel with that of the IP(3) system during maturation of oocytes. Sea urchin oocytes were cultivated in vitro and Ca(2+) transients induced by photolysis of caged IP(3) or caged cADPR were measured during maturation. Oocytes spontaneously began to maturate in seawater. More than 50% of oocytes underwent germinal vesicle breakdown within 25 h and the second meiosis within 35 h, but it took more than 24 h until they became functionally identical to in vivo-matured eggs. Both IP(3) and cADPR induced Ca(2+) transients comparable to those of in vivo-matured eggs later than 24 h from the second meiosis. However, cADPR induced a small Ca(2+) transient even before meiosis, whereas IP(3) and sperm almost did not.     

A rapid change in phosphorylation on tyrosine accompanies fertilization of sea urchin eggs.

Alterations in protein phosphorylation, particularly phosphorylation on tyrosine, frequently accompany cell change and are important agents in the cascades initiated by extracellular signals. This paper examines whether the activation of the sea urchin egg at fertilization involves an early and rapid phosphotyrosine response. Using an anti-phosphotyrosine antibody and a rapid sampling technique, we find a very early increase in the phosphorylation on tyrosine of two proteins of approximately 91 kDa and 138 kDa. A similar phosphorylation occurs after activation of the eggs by the calcium ionophore, ionomycin, suggesting the stimulation of a Ca(2+)-sensitive pathway. The timing and Ca2+ sensitivity suggest a role in the primary signal transduction events of fertilization.     

Cloning and characterization of a phospholipase C-beta isoform from the sea urchin Lytechinus pictus

Calcium is a ubiquitous intracellular signaling molecule controlling a wide array of cellular processes including fertilization and egg activation. The mechanism for triggering intracellular Ca(2+) release in sea urchin eggs during fertilization is the generation of inositol-1,4,5-trisphosphate by phospholipase C (PLC) hydrolysis of phosphatidylinositol-4,5-bisphosphate. Of the five PLC isoforms identified in mammals (beta, gamma, delta, epsilon and zeta), only PLCgamma and PLCdelta have been detected in echinoderms. Here, we provide direct evidence of the presence of a PLCbeta isoform, named suPLCbeta, within sea urchin eggs. The coding sequence was cloned from eggs of Lytechinus pictus and determined to have the greatest degree of homology and identity with the mammalian PLCbeta4. The presence of suPLCbeta within the egg was verified using a specifically generated antibody. The majority of the enzyme is localized in the non-soluble fraction, presumably the plasma membrane of the unfertilized egg. This distribution remains unchanged 1 min postfertilization. Unlike PLCbeta4, suPLCbeta is activated by G protein betagamma subunits, and this activity is Ca(2+)-dependent. In contrast to all known PLCbeta enzymes, suPLCbeta is not activated by Galphaq-GTPgammaS subunit suggesting other protein regulators may be present in sea urchin eggs.     

Distinct specificity in the recognition of phosphoinositides by the pleckstrin homology domains of dynamin and Bruton's tyrosine kinase.

Pleckstrin homology (PH) domains may act as membrane localization modules through specific interactions with phosphoinositide phospholipids. These interactions could represent responses to second messengers, with scope for regulation by soluble inositol polyphosphates. A biosensor-based assay was used here to probe interactions between PH domains and unilamellar liposomes containing different phospholipids and to demonstrate specificity for distinct phosphoinositides. The dynamin PH domain specifically interacted with liposomes containing phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] and, more weakly, with liposomes containing phosphatidylinositol-4-phosphate [PI(4)P]. This correlates with phosphoinositide activation of the dynamin GTPase. The functional GTPase of a dynamin mutant lacking the PH domain, however, cannot be activated by PI(4,5)P2. The phosphoinositide-PH domain interaction can be abolished selectively by point mutations in the putative binding pocket predicted by molecular modelling and NMR spectroscopy. In contrast, the Bruton's tyrosine kinase (Btk)PH domain specifically bound liposomes containing phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3]: an interaction requiring Arg28, a residue found to be mutated in some X-linked agammaglobulinaemia patients. A rational explanation for these different specificities is proposed through modelling of candidate binding pockets and is supported by NMR spectroscopy.     

A synthetic peptide of the pseudosubstrate domain of protein kinase C blocks cytoplasmic alkalinization during activation of the sea urchin egg

Multiple second messenger pathways have been proposed for transduction of the sperm-egg fusion event during fertilization of sea urchin eggs. Cytoplasmic alkalinization due to increased Na(+)-H+ antiport has been causally linked to many of the metabolic events during fertilization. Two possible second messenger pathways coupling sperm-egg fusion and antiporter activity are activation of protein kinase C (PKC) and Ca2(+)-calmodulin kinase. A selective inhibitor of PKC is PKC(19-36), a synthetic peptide of the pseudosubstrate domain of the kinase. Injection of PKC(19-36) into unfertilized sea urchin eggs blocked cytoplasmic alkalinization during activation by phorbol 12-myristate 13-acetate, a PKC agonist. The rise in pH during fertilization was partially blocked by PKC(19-36), which suggested that multiple pathways regulate the antiporter during fertilization. The use of fluorescein chromophores to measure intracellular pH in sea urchin eggs is also discussed.     

Phosphoinositide metabolism at fertilization of sea urchin eggs measured with a GFP-probe

Fertilization elicits a dramatic, transient rise in Ca2+ within the egg which is an essential component of egg activation and consequent initiation of development. In the sea urchin egg, three distinct Ca2+ stores have been identified which could, either individually or in combination, initiate Ca2+ release at fertilization. Inositol 1,4,5-trisphosphate (IP3) production by phospholipase C (PLC) has been suggested as the singular signal in initiating the Ca2+ transient. Other studies indicate that Ca2+ stores gated by cyclic adenosine diphosphate ribose (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP) are also necessary. We have examined the temporal relationship between the Ca2+ rise and IP3 production at fertilization in vivo within individual eggs using a green fluorescent protein (GFP) coupled to a pleckstrin homology (PH) domain that can detect changes in IP3. Translocation of the probe occurred after the Ca2+ rise was initiated. Earlier, and possibly smaller, IP3 changes could not be excluded due to limitations in probe sensitivity. High IP3 levels are maintained during the decline in cytoplasmic Ca2+, suggesting that later IP3 metabolism might not be related to regulation of Ca2+, but may function to modulate other PIP2 regulated events such as actin polymerization or reflect other novel phosphoinositide signaling pathways.     

Calcium pools in sea urchin eggs: roles of endoplasmic reticulum and mitochondria in relation to fertilization.

A preparation of sea urchin eggs permeabilized with digitonin (40 microM for 2.5 min) was used to study the kinetic characteristics of the two cellular compartments suspected to play a key role in cellular calcium transfer during fertilization: an ATP-dependent Ca2+ pool (Km = 0.47 microM; Vm = 0.48 nmol/min.mg protein) probably located in the endoplasmic reticulum and a mitochondrial Ca2+ pool (Km = 1.50 microM; Vm = 0.12 nmol/min.mg protein). Fertilization triggered a decrease in the rate of ATP dependent uptake by the non-mitochondrial pool (Km = 0.59 microM; Vm = 0.15 nmol/min.mg protein) while it transiently increased the Ca2+ uptake into mitochondria (2 min post-fertilization: Km = 2.20 microM; Vm = 0.40 nmol/min.mg protein). Microanalysis studies performed on quickly frozen, freeze substituted and embedded eggs showed a transient Ca2+ enrichment of mitochondria soon after fertilization thus suggesting that mitochondria behave as a Ca2+ sink at fertilization. Results are discussed in relation to the role of endoplasmic reticulum and mitochondria in handling free calcium during the early period following sea urchin egg fertilization.     

Protein tyrosine kinase-dependent release of intracellular calcium in the sea urchin egg

The aminoguanide, methylglyoxal bis(guanylhydrazone) (MGBG), was shown to stimulate phosphorylation of RR-SRC, a synthetic protein tyrosine kinase (PTK) substrate, and different levels of tyrosyl phosphorylation of endogenous proteins in a sea urchin egg membrane-cortex preparation. Stimulating protein tyrosine kinase activity in the sea urchin egg stimulated intracellular Ca2+ release, because microinjection of 1-5 mM of MGBG into unfertilized eggs triggered a transient rise in intracellular Ca2+ activity ([Ca2+]i) after a brief latent period. Pretreating eggs with PTK-specific inhibitors, genistein or tyrphostin B42, significantly inhibited the MGBG-induced rise in [Ca2+]i. Methylglyoxal bis(guanylhydrazone) stimulation of PTK activities in the unfertilized sea urchin egg appeared to trigger Ca2+ release through phospholipase C (PLC)-dependent inositol 1,4,5-trisphosphate (InsP3) production. The MGBG-induced Ca2+ response could be suppressed in eggs preloaded with the InsP3 receptor antagonist, heparin, and was reduced in eggs pretreated with U73122, a PLC inhibitor. However, the response was unchanged in eggs treated with nicotinamide, an inhibitor of ADP-ribosyl cyclase, or nifedipine, an inhibitor of nicotinic acid adenine dinucleotide phosphate activity. These results suggest that MGBG may be useful as a chemical agonist of PTK in sea urchin eggs and allow direct testing of the PTK requirement for the transient rise in [Ca2+]i in sea urchin eggs during fertilization. Although genistein was observed to significantly delay the onset, the sperm-induced Ca2+ response in PTK inhibitor-loaded eggs otherwise appeared normal. Therefore, it was concluded that sea urchin eggs contain a PTK-dependent pathway that can mediate intracellular Ca2+ release, but PTK activity does not appear to be required for the fertilization response.     

Synergistic release of calcium in sea urchin eggs by caffeine and ryanodine.

A transient rise in intracellular Ca2+ during fertilization is necessary for activation of the quiescent sea urchin egg. Several mechanisms contribute to the rise in Ca2+ including influx across the egg plasma membrane and release from intracellular stores. The egg contains both IP3-sensitive and -insensitive Ca2+ release mechanisms and in this study we have used single-cell spectrofluorimetry to examine the effects of caffeine and ryanodine on Ca2+ release in eggs preloaded with fura 2. Caffeine induced a small Ca2+ release that was insensitive to heparin or ruthenium red. Ca2+ liberation by caffeine could be augmented by prior treatment with thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ ATPase. Variable Ca2+ releases were observed in response to microinjection of ryanodine. The action of ryanodine appeared to be enhanced by prior injection of heparin and partially inhibited by ruthenium red. The release of Ca2+ by caffeine or ryanodine was generally insufficient to trigger cortical granule exocytosis, thus these eggs could be fertilized and a second Ca2+ release during fertilization was measured. Unlike the caffeine- and ryanodine-sensitive Ca(2+)-induced Ca2+ release mechanism in somatic cells, the graded responses in eggs suggested this caffeine- and ryanodine-sensitive release mechanism is not sensitive to sudden changes in Ca2+. Thus we could examine the combined actions of caffeine and ryanodine on Ca2+ release, which were synergistic. Caffeine treatment of ryanodine-injected eggs or ryanodine injection of caffeine-treated eggs stimulated a Ca2+ release significantly larger than the release by either drug independently. The experiments presented here suggest that sea urchin eggs liberate Ca2+ in response to caffeine and ryanodine; however, the regulation of this release differs from that described for caffeine- and ryanodine-sensitive Ca(2+)-induced Ca2+ release of somatic cells.     

Mastoparan induces Ca2+-independent cortical granule exocytosis in sea urchin eggs

In most species, cortical granule exocytosis is characteristic of egg activation by sperm. It is a Ca(2+)-mediated event which results in elevation of the vitelline coat to block permanently the polyspermy at fertilization. We examined the effect of mastoparan, an activator of G-proteins, on the sea urchin egg activation. Mastoparan was able to induce, in a concentration-dependent manner, the egg cortical granule exocytosis; mastoparan-17, an inactive analogue of mastoparan, had no effect. Mastoparan, but not sperm, induced cortical granule exocytosis in eggs preloaded with BAPTA, a Ca(2+) chelator. In isolated egg cortical lawns, which are vitelline layers and membrane fragments with endogenously docked cortical granules, mastoparan induced cortical granule fusion in a Ca(2+)-independent manner. By contrast, mastoparan-17 did not trigger fusion. We conclude that in sea urchin eggs mastoparan stimulates exocytosis at a Ca(2+)-independent late site of the signaling pathway that culminates in cortical granule discharge.     

Extracellular Ca2+ influx is crucial for the early embryonic development of the sea urchin Echinometra lucunter

The involvement of Ca(2+) in the activation of eggs and in the first steps of the embryonic development of several species is a well-known phenomenon. An association between Ca(2+) sources with the fate of the blastopore during embryonic development has been investigated by several authors. Ca(2+) influx mediated by voltage-gated channels and Ca(2+) mobilization from intracellular stores are the major sources of Ca(2+) to egg activation and succeeding cell divisions. Studies on sea urchins embryonic development show that intracellular Ca(2+) stores are responsible for egg activation and early embryogenesis. In the present work we investigated the involvement of extracellular Ca(2+) in the first stages of the embryonic development of the sea urchin Echinometra lucunter. Divalent cation chelators EDTA and EGTA strongly blocked the early embryonic development. Adding to this, we demonstrated the involvement of voltage-gated Ca(2+) channels in E. lucunter embryogenesis since Ca(2+) channel blockers powerfully inhibited the early embryonic development. Our data also revealed that Ca(2+) influx is crucial for embryonic development during only the first 40?min postfertilization. However, intracellular Ca(2+) remains mandatory to embryonic development 40?min postfertilization, seen that both the intracellular Ca(2+) chelator BAPTA-AM and calmodulin antagonists trifluoperazine and chlorpromazine inhibited the first stages of development when added to embryos culture 50?min postfertilization. Our work highlights the crucial role of extracellular Ca(2+) influx through voltage-gated Ca(2+) channels for the early embryonic development of the sea urchin E. lucunter and characterizes an exception in the phylum Echinodermata.